Carboxyl-terminal and intracellular loop sites for CRF1 receptor phosphorylation and β-arrestin-2 recruitment:: a mechanism regulating stress and anxiety responses

The primary goal was to test the hypothesis that agonist-induced corticotropin-releasing factor type 1 (CRF1) receptor phosphorylation is required for beta-arrestins to translocate from cytosol to the cell membrane. We also sought to determine the relative importance to beta-arrestin recruitment of motifs in the CRF1 receptor carboxyl terminus and third intracellular loop. beta-Arrestin-2 translocated significantly more rapidly than beta-arrestin-1 to agonist-activated membrane CRF1 receptors in multiple cell lines. Although CRF1 receptors internalized with agonist treatment, neither arrestin isoform trafficked with the receptor inside the cell, indicating that CRF1 receptor-arrestin complexes dissociate at or near the cell membrane. Both arrestin and clathrin-dependent mechanisms were involved in CRF1 receptor internalization. To investigate molecular determinants mediating the robust beta-arrestin-2-CRF1, receptor interaction, mutagenesis was performed to remove potential G protein-coupled receptor kinase phosphorylation sites. Truncating the CRF1 receptor carboxyl terminus at serine-386 greatly reduced agonist-dependent phosphorylation but only partially impaired beta-arrestin-2 recruitment. Removal of a serine/threonine cluster in the third intracellular loop also significantly reduced CRF1 receptor phosphorylation but did not alter beta-arrestin-2 recruitment. Phosphorylation was abolished in a CRF1 receptor possessing both mutations. Surprisingly, this mutant still recruited beta-arrestin-2. These mutations did not alter membrane expression or cAMP signaling of CRF1 receptors. Our data reveal the involvement of at least the following two distinct receptor regions in beta-arrestin-2 recruitment: 1) a carboxyl-terminal motif in which serine/threonine residues must be phosphorylated and 2) an intracellular loop motif configured by agonist-induced changes in CRF1 receptor conformation. Deficient beta-arrestin-2-CRF1 receptor interactions could contribute to the pathophysiology of affective disorders by inducing excessive CRF1 receptor signaling.